Made available in DSpace on 2016-04-15T12:59:57Z (GMT). No. of bitstreams: 2 marco_stimamiglio_ioc_dout_2009.pdf: 7045829 bytes, checksum: 6668ee161093cf29f56e28a841129af4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A colonização do timo por precursores hematopoéticos representa um evento crucial para o desenvolvimento deste próprio órgão, assim como garante a diferenciação e a formação do repertório de células T maduras. Entretanto, os mecanismos moleculares que dirigem este processo não são totalmente conhecidos. A entrada destes precursores depende da ativação de uma cascata de sinalizações intermoleculares, onde participam algumas moléculas, como as integrinas e as quimiocinas. Os receptores Eph, que compõem a maior família de receptores tirosina-quinase, representam importantes moléculas reguladoras do desenvolvimento de sistemas e órgãos, sendo encontrados também no tecido linfóide. Mais recentemente, essa família de receptores, juntamente com seus ligantes, efrinas, foi descrita como moléculas co-estimulatórias de sinais transmitidos em linfócitos T pelo receptor de antígeno, por quimiocinas e integrinas. Neste contexto, o objetivo central deste trabalho foi o de avaliar as possíveis funções dos receptores Eph, em particular EphB2, em modular a atividade migratória de precursores T durante os processos de colonização do timo e maturação intratímica de linfócitos Nossos resultados demonstram a expressão dos receptores EphB2 no timo de camundongos e a sua participação tanto nos processos iniciais da organogênese do timo, quanto na diferenciação intratímica de timócitos. Este receptor, assim como seus principais ligantes, também é expresso em células precursoras derivadas da medula óssea de camundongos e é capaz de modular a migração e a capacidade de entrada destes precursores em lóbulos tímicos alinfóides. Além disso, vimos que a falta deste receptor, ou de seu domínio catalítico tirosina-quinase, promove uma redução na deposição de proteínas da matriz extracelular e de quimiocinas no timo, assim como resulta em importante inibição da entrada dos precursores hematopoiéticos neste órgão. De igual maneira, o desequilíbrio dos sinais transmitidos pelo complexo EphB2/efrina-B impede o correto posicionamento intratímico destes precursores, possivelmente levando a um bloqueio na maturação dos timócitos. Finalmente, demonstramos que a ausência do receptor ou dos sinais EphB2 não modifica os níveis de expressão de outros receptores como integrinas e receptores de quimiocina nos precursores hematopoiéticos e timócitos, mas possivelmente modula sua atividade e, desta forma, a atividade migratória destas células frente a estímulos hapto e quimiotáticos. Em conjunto, nossos resultados apontam uma importante participação dos sinais desencadeados pelo complexo Eph/efrina e sua co-regulação com outros receptores que modulam o processo de migração dos precursores de células T, desde sua entrada no timo, até o seu correto desenvolvimento e migração dentro deste órgão / Thymus settling by hematopoietic progenitors represents a crucial event during thymus ontogeny and guarantees the proper differentiation of the T-cell repertoire. However, the molecular mechanisms that drive such process are not completely understood. Progenitor settling depends on the activation of intercellular signaling cascades, where some integrins and chemokines play a role. Eph receptors, th e major tyrosine-kinase receptor family, are important regulatory molecules for the development of several systems and organs, being also expressed in lymphoid tissues. More recently, this receptor family, conjointly with the corresponding ligands, the ephrins, has been reported as costimulatory molecules for the T- cell receptor, chemokine receptors and integrins on T lymphocytes. In this context, the aim of this work was to evaluate the possible functions of Eph receptors, in particular EphB2, as modulators of T-cell progenitor migration during thymus settling and intrathymic T-cell maturation. Our results demonstrate that EphB2 receptors are expressed in the mouse thymus and participate in its organogenesis and intrathymic T-cell development. This receptor and its main ligands are also expressed in mouse bone marrow-derived progenitor cells, being able to modulate migration and the ability of these cells to settling thymic lobes. Moreover, the lack of such receptor, or its tyrosine-kinase domain, results in a reduced deposition of extracellular matrix proteins and chemokines in the thymus, and leads to an important inhibition of thymus settling by hematopoietic progenitors. Furthermore, an imbalance of the signals transmitted by EphB2/ephrin-B complex prevents proper intrathymic positioning of progenitor cells, possibly causing a blockade in thymocyte maturation. Finally, we demonstrated that the lack of EphB2 receptor or signaling does not change the expression level of integrins and chemokine receptors on hematopoietic progenitors and thymocytes, but possibly modulates the activity of these receptors and the cell migration activity through hapto and chemotactic stimuli. Taken together, our results point to an important participation of Eph/ephrin complex signaling and its cross-regulation with other receptors that modulates T-cell migration process, from thymus settling until the pr oper thymocyte development within the organ.

The use of highly active antiretroviral therapy (HAART) has significantly decreased the mortality rate of HIV-1 patients, however the increased survival has led to the development of complications associated with the persistence of the viral infection. Nearly half of HIV-1-infected individuals develop HIV-associated neurocognitive disorders (HAND) as the effects of the chronic infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxicity of HIV-1 has largely been attributed to the inflammation caused by viral replication and the altered signaling of astrocytes, microglia, and macrophages. Although HAART has improved the control of viral replication, the effects from inflammation remain a concern, particularly those of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α has been a therapeutic target for other diseases associated with chronic inflammation, such as rheumatoid arthritis, but emerging evidence has suggested that TNF-α signaling can have a dual role, especially in the CNS, proving the complexity in the modulation of the TNF-α pathway. Although the detrimental effects of TNF-α have been well-characterized, we lack a complete understanding of the beneficial role of TNF-α. TNF-α signaling has largely been considered to be neurotoxic but has been able to regulate neurite outgrowth in the context of neural development. Since TNF-α is upregulated in various neurodegenerative conditions, we considered potential outcomes of TNF-α on neurite outgrowth following injury. Initially, most would assume that TNF-α would prevent neurite outgrowth as apoptosis is a common outcome of TNF-α-induced signaling. If TNF-α signaling strictly prevents neurite outgrowth, anti-TNFα therapies could be considered to reverse this effect. However, upon induced injury, we observed an increase in neurite regrowth following induced injury in human primary fetal neurons, demonstrating a strong need for a deeper understanding of this dual role of TNF-α. Anti-TNF-α therapies have been considered for HIV-1-infected patients to reduce the chronic inflammation, however inhibiting TNF-α signaling could have side-effects that could prevent neuronal recovery from HIV-1 effects. Targeting pathways downstream of TNF-α signaling would be more advantageous to mediate the beneficial role of TNF-α in the CNS. We investigated the transcriptional effects of TNF-α treatment on neurons to uncover a potential pathway to promote neurite outgrowth. One pathway we have discovered to be beneficial in primary human fetal neurons is TNF-α-induced Ephrin B2 upregulation. Ephrin B2 (EphB2) receptors are important mediators of neuronal development and synaptic plasticity, however little has been established in regards to their role in HIV and inflammation, particularly in the CNS. EphB2 can mediate axonal development by providing retractive cues to assist the axon to reach the target, but EphB2 can also promote dendritic branching to improve learning and memory, which would be particularly beneficial for HAND patients that experience cognitive deficits. We observed a correlation between the upregulation of EphB2 in response to TNF-α and neurite outgrowth, which provides a potential pathway to repair damaged neurons and re-establish lost neuronal connections. Dendritic pruning and neuronal loss has been observed in HAND patients, so this ability to promote repair could prevent, improve, or recover the cognitive deficits experienced by HIV-patients with HAND. TNF-α, although primarily known to induce neurotoxicity, strongly activates the nuclear factor-kappaB (NF-κB) pathway, which can have a very wide range of transcriptional effects. Therefore, our hypothesis is that the TNF-α-induced neurite regrowth occurs through an upregulation EphB2 in an NF-κB-dependent pathway. TNF-α has been well established to induce NF-κB signaling, mostly by promoting the translocation of the NF-κB p65 DNA binding factor to the nucleus for transcriptional regulatory effects. NF-κB can regulate neuronal growth and process development of both dendrites and axons, which would correlate to the neurite regrowth observed following TNF-α upon induced injury. The regulation of EphB2 by NF-κB has not been extensively studied, but EphB2 can be negatively regulated by an NF-κB family member, c-Rel. We analyzed the EphB2 promoter and identified three NF-κB p65 binding sites upstream from the transcriptional start site, which provided insight to our hypothesis. We established that p65 directly binds to and can regulate EphB2 promoter activity in response to TNF-α. Since the dual role of TNF-α can be dependent on the receptor through which the signaling proceeds, either TNF-α receptor 1 (TNFR1) or TNF-α receptor 1 (TNFR2), we investigated if this upregulation of EphB2 is receptor dependent and determined EphB2 is induced primarily through activation of TNFR2. Neurons express both receptors, however, the effects of TNF-α to promote neuroprotection and repair primarily occur through the TNF-α/TNFR2 regulatory axis. Although we have been established the mechanism of TNF-α-induced EphB2 and there is a strong correlation with neurite outgrowth following induced injury, we considered the possibilities to modulate EphB2 in the absence of TNF-α to demonstrate the direct effects of EphB2 expression. Several approaches could be used to mediate EphB2 activation or inhibition in vitro. RNA interfering techniques, such as small interfering RNA (siRNA), are useful, but we were interested in a complete knockout strategy. Since our approach was to assess the effects of EphB2 knockout only on neurite outgrowth following induced injury, a knockout animal model would not be appropriate, as a lack of EphB2 would affect the development of the neurons, unless an inducible knockout model was established. This is a lengthy and elaborate process and, more importantly, would only be available in a non-human model. Other techniques, such as transcription activator like effector nucleases (TALENs), can generate knockout systems that are targeted to specific regions of a gene, but specific binding proteins must be created to recruit the endonucleases to the target. Clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) has emerged as a specific and relatively easy technique to knockout genes of interest and uses short RNA sequences to guide Cas9 endonucleases to target regions to create double stranded breaks in the DNA to silence the gene. Once concern with Cas9 is specificity to target only the desired region of the gene, as off-target effects can occur and may result in unwanted gene silencing. A Cas9 mutant, Cas9 nickase (Cas9n), has been created to have more specificity by requiring two guide RNAs to recruit two Cas9 nickases to generate a double stranded break as they function as nickases to only create a nick in one DNA strand. We developed this strategy to remove exon 1 of the EphB2 gene by using two pairs of Cas9 nickases, with four guide RNAs, to eliminate any chance for off-target effects but retaining the desired outcome of and EphB2 knockout. We validated the system by demonstrating that a knockout of EphB2 increases adhesion and prevents migration in human embryonic kidney 293T (HEK293T) cells. Although this cell model is not a neuronal cell model, the migration assay demonstrates the functional loss of EphB2. We also created an inducible Ephb2 system to overexpress EphB2. Together these provide essential tools to verify the direct involvement of EphB2 in neurite outgrowth. Taken together, our studies characterize a novel mechanism for neurite outgrowth following injury in neurons: TNF-α/TNFR2-induced EphB2 signaling in an NF-κB p65-dependent manner. In addition to the established mechanism, we developed a technique to assess the effects of EphB2 knockout and overexpression in the context of neurite outgrowth: EphB2-targeted-Cas9n and EphB2 inducible construct. This mechanism yields insight into a potential downstream pathway to be utilized to repair damaged regions in the brain and reverse cognitive deficits in neurodegenerative conditions, especially in a chronic inflammatory environment, such as HIV-1 infection. The strategies created provide a valuable toolset to demonstrate the direct effects of modulating EphB2 signaling, not only in neurons for effects on neuronal health and synaptic plasticity, but also in other disease models, such as glioblastoma, in which EphB2 was demonstrated to promote invasion and migration of tumor cells. These observations and the usefulness of the modulatory strategies likely extend to multiple neurodegenerative diseases that demonstrate cognitive deficits that correlate to neuroinflammation. / Biomedical Neuroscience

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 3 uppsatser.

Research has identified a linear relationship between saturated fat intake and colon cancer, and has demonstrated that high fat diets enhance tumorigenesis through elevation of secondary bile acids such as deoxycholic acid (DCA). We and others have shown that DCA can manipulate cell adhesion by decreasing expression of E-cadherin and increasing expression of beta-catenin. We have also shown that DCA significantly reduces EphB2 expression, which regulates cell positioning and segregation. Importantly, vitamin D can reinstate membranous E-cadherin/beta-catenin interactions and increase E-cadherin expression. In the present study, we sought to analyze the effects of DCA and vitamin D (cholecalciferol) treatment on EphB2 in colorectal cancer cells. Pre-treatment with cholecalciferol restored EphB2 expression in a dose-dependent manner, even with combined DCA treatment. This observation may be EGFR-dependent, suggesting that cholecalciferol may antagonize the effects of DCA. Taken together, these results suggest that cholecalciferol may represent an adjuvant therapy for colorectal cancer patients.

The underlying mechanism of morphine tolerance remains unclear. EphB2 regulates synaptic efficiency with respect to learning and memory. Previously, we demonstrated that loss of EphB2 significantly accelerates the rate of morphine tolerance and alters behavioural responses to morphine following tolerance. However, EphB2 null mice exhibit no significant alteration in their metabolism of morphine compared to littermate controls, or altered mu opioid receptor expression levels within the spinal cord or brain compared to littermate controls. Therefore, we investigated whether loss of EphB2 alters learned responsiveness to morphine through modification of hippocampal function. Interestingly, results indicate that electrolytic lesions of the dorsal hippocampus of wild-type mice display similar behavioural responses seen in EphB2 null mice compared to sham operated controls. These findings suggest that loss of EphB2 function within the hippocampus is a critical feature in mediating morphine-dependent tolerance, and suggests a novel role for EphB2 receptor signaling in opiate-dependent learning.

The underlying mechanism of morphine tolerance remains unclear. EphB2 regulates synaptic efficiency with respect to learning and memory. Previously, we demonstrated that loss of EphB2 significantly accelerates the rate of morphine tolerance and alters behavioural responses to morphine following tolerance. However, EphB2 null mice exhibit no significant alteration in their metabolism of morphine compared to littermate controls, or altered mu opioid receptor expression levels within the spinal cord or brain compared to littermate controls. Therefore, we investigated whether loss of EphB2 alters learned responsiveness to morphine through modification of hippocampal function. Interestingly, results indicate that electrolytic lesions of the dorsal hippocampus of wild-type mice display similar behavioural responses seen in EphB2 null mice compared to sham operated controls. These findings suggest that loss of EphB2 function within the hippocampus is a critical feature in mediating morphine-dependent tolerance, and suggests a novel role for EphB2 receptor signaling in opiate-dependent learning.

No description available.

Eph receptors are known to be overexpressed in various types of cancer and are therefore promising targets for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of Eph-overexpressing tumors, monitoring responses to therapy directed toward Eph, and thus improvement in patient outcomes. This work report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW and SWLAY amino acid motif, with high affinity for the EphA2 and B2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphA2- and EphB2-antagonistic peptides were varied at the C-terminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [18F]AFP and [18F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [18F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

Eph receptors are known to be overexpressed in various types of cancer and are therefore promising targets for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of Eph-overexpressing tumors, monitoring responses to therapy directed toward Eph, and thus improvement in patient outcomes. This work report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW and SWLAY amino acid motif, with high affinity for the EphA2 and B2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphA2- and EphB2-antagonistic peptides were varied at the C-terminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [18F]AFP and [18F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [18F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

No description available.